Enzyme-catalyzed reactions often have the advantages of proceeding with great chemical specificity under relatively mild conditions, and often accomplish what man finds difficult, if not impossible, to duplicate in the laboratory. For such reasons there is increasing emphasis on the use of enzymatic processes on a commercial scale. One example, of many which could be cited, is the conversion of glucose to fructose using glucose isomerase.
Enzymes are water soluble, and if they are merely used in aqueous solutions recovery of enzyme for reuse is difficult and expensive. Using the enzyme only once affords a process which is relatively expensive. Consequently, many techniques have been developed for immobilizing the enzyme in such a way that substantial enzymatic activity is displayed while the enzyme itself remains rigidly attached to some water-insoluble support, thereby permitting reuse of the enzyme over substantial periods of time and for substantial amounts of feedstock. One illustration of a method for immobilizing an enzyme is found in Levy and Fusee, U.S. Pat. No. 4,141,857, where a polyamine is adsorbed on a metal oxide such as alumina, treated with an excess of a bifunctional reagent, such as glutaraldehyde, so as to cross-link the amine, thereby entrapping the resulting polymer in the pores of the metal oxide, and then contacting the mass with enzyme to form covalent bonds between the pendant aldehyde groups and an amino group on the enzyme.
The useful life of an immobilized enzyme system is limited by a continual decrease in enzymatic activity. Among the many mechanisms which lead to enzyme deactivation in such systems are: poisoning of the enzymes by impurities in the feedstock; other chemical modification of the enzyme during its utilization; denaturation of the enzyme; rupture of the bond between the pendant group and the enzyme leading to dissolution of the enzyme; cleavage of the bond between the pendant group and the intermediate binding layer; loss of the binding layer as, for example, by physical ablation or cleavage of the chemical bonds which hold it to the support.
Whatever the mechanism of the enzyme deactivation, reactivation of a deactivated immobilized enzyme system would prove to be a substantial advance in the art as well as being economically highly desirable. At least conceptually, two distinct approaches to reactivation are possible. One mode would be to rejuvenate the enzyme itself, i.e., assuming no physical loss of enzyme, the transformations which rendered it inactive would be reversed and the enzyme would revert to its initial active state. The alternative is to restore the immobilized enzyme system to that state initially present immediately prior to attachment of enzyme, so that it would be capable of binding fresh, active enzyme once again. This invention relates to the latter approach.